Given that the human mucosae is 200-400 mm thick, and the number of mucosal pathogens is large, understanding the potential protective mechanisms of the host at these complex mucosal tissue sites is a priority for the generation of efficacious vaccines. One strategy for the generation of more effective vaccines is to understand the host mucosal immune response to these organisms. Towards that end, the long-range goal of our research program is to understand mechanisms of mucosal immunity in the host response to enteric pathogens, with the overall objective of reducing the morbidity and mortality in these diseases. Our central hypothesis is that the FAE plays a dynamic primary role in the earliest stages of mucosal innate immune responses to enteric pathogens, and this role is driven by soluble mediators and mucosal cell-cell interactions. The objective of the current application is to investigate the premise that the FAE contributes to innate mucosal immunity through the elicitation of an early host response that directly affects the functional capacity of immature DC and macrophages in the sub-epithelial dome. Two Aims will test specific aspects of our central hypothesis: Specific Aim 1: to determine the potential of pathogen-stimulated FAE to secrete apically-derived soluble mediators and analyze the relevance of these molecules to host mucosal immune reactions. After the FAE model is optimized, the capacity of these cells to secrete anti- inflammatory factors, acute phase reactants, pro-inflammatory cytokines, and chemokines will be assessed after bacterial infection by reverse transcriptase-polymerase chain reaction (RT-PCR), ribonuclear protection assays (RPA), and complementary deoxyribose nucleic acid (cDNA) microarray techniques. The ability of pathogen-induced FAE to secrete anti-microbial factors that reduce bacterial load or protect immune cells also will be evaluated. Specific Aim 2: to analyze the cellular mechanism(s) by which sub- epithelial macrophages and immature DC interact with the pathogen-stimulated FAE. Transmigration of macrophages to the apical surface of pathogen-induced model FAE will be evaluated, as will the capacity of immature DC to mature after exposure to the pathogen-induced model FAE. We have developed an in vitro model of the human small intestine PP dome that contains M cells, IEC, macrophages, and immature DC to study the early steps in the generation of a mucosal immune response. This model is the first that permits a dissection of the functional capacity of the FAE and analysis of cross-talk of these cells with two other mucosal innate cell types. Since interventions that focus on early steps in the process would be the most desirable, our studies of the initial cell types involved in this complex set of reactions should provide data applicable to: (1) determining the underlying cellular and molecular mechanisms of mucosal innate immunity; and (2) the development of successful therapeutic interventions.